The proposed project is a Phase I STTR collaboration between Vala Sciences Inc, a small business that specializes in the development of cell-based high content/high throughput assays, and the Sanford Burnham Medical Research Institute, a worldwide leader in biomedical research. The project will develop an assay (Kinetic Image Cytometer-Neuron, or KIC-Neuron) to quantify the effect of test compounds on neuronal transmission and the development of neuronal cell circuits utilizing optogenetic methods. For KIC-Neuron, primary rat hippocampal neurons will be cultured in a high throughput format (96 or 384 well), then transduced with lenti-viruses encoding light-activated channel rhodopsin, and loaded with fluorescent indicators of DNA (to visualize the nuclei) and intracellular calcium. This will enable depolarization of the cells via use of a flash of light, which leads to neurotransmission across the neuronal cultures and elevation of intracellular calcium in post-synaptic neurons, which is quantified via automated microscopy and image analysis techniques. Following the activity assay, the cells are also fixed, labeled, and imaged for neuronal biomarkers, which can be correlated on a cell-by-cell basis to neuroactivity. The assay system will be used to quantify the effects of short -term exposure to compounds (such as the presence of ion channel blockers or antagonists of neural transmitter receptors during the activity assay), and also to quantify potential long-term effects of compounds on the neural circuits. The latter phenomenon is relevant to the Chemo-brain condition, which is a loss cognitive ability that develops in cancer patients following chemotherapy, and may be related to other forms of memory loss and dementia, as well, including Alzheimer's. The assay will be of interest to government agencies including the US Environmental Protection Agency, and NIH institutes (NCI and NHCLBI) as well as to both pharmaceutical and academic researchers. KIC-NEURON will be commercialized for Vala's contract research screening as well as developed into a product line featuring kits, software, and instrumentation.